Convenient twice daily foscarnet in induction therapy of AIDS-associated cytomegalovirus retinitis. The Foscarnet Italian Study Group.
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AIDS-Related Opportunistic Infections↗
Biomedical subjects
G Gerna
Publications and source records attributed to G Gerna.
Use of reverse-transcription polymerase chain reaction for detection of rubella virus RNA in cell cultures inoculated with clinical samples.
A recently developed reverse transcription-nested polymerase chain reaction (RT-nPCR) method for rubella virus (RV) RNA detection was assessed in a series of African green monkey kidney (AGMK) cell cultures inoculated with clinical samples from patients with suspected RV infection. Results were compared with those of conventional virus isolation/identification. The assay included an internal control of amplification consisting of a synthetic RNA molecule mimicking the RV E1 target sequence. A semiquantitation of RV RNA was achieved by comparing the relative band intensity of internal control and RV E1 fragment. RT-nPCR was positive in 15/16 (94%) RV isolation-positive cultures and in 12/60 (20%) RV isolation-negative cultures. All 27 cell cultures positive by RT-nPCR had been inoculated with clinical samples taken from patients with ascertained RV infection or given RV vaccination and consisted of cells harvested 1-10 days after primary inoculation of clinical samples. No RV RNA was found in any of the cell cultures inoculated with 14 clinical samples from 6 patients in whom RV infection was excluded. When considering the time-course of RV infection, it was found that RV RNA could be detected as early as 4 days p.i. in 10/21 (48%), and by 7-10 days p.i. in 27/28 (96%) cell cultures, whereas by the same time RV was isolated in only 7/16 (44%) cell cultures. Semiquantitation showed that: i) viral RNA amount progressively increased with time; ii) cell cultures containing very low levels of viral RNA one week p.i. either required a few blind passages for virus recovery or remained negative for RV isolation. Finally, PCR inhibitors were found in 10/164 (6%) cell cultures examined. In conclusion, RT-nPCR proved to be very sensitive and very specific and greatly reduced RV detection time in inoculated cell cultures.
Adult↗
Anticytomegalovirus T-cell clones.
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Bone Marrow Transplantation↗
Diagnosis of human cytomegalovirus infections in the immunocompromised host.
BACKGROUND: In the last decade several major advances have been made in the rapid diagnosis of human cytomegalovirus (HCMV) infections and disease in immunocompromised patients both at the immunological and molecular level. OBJECTIVES: The objective was to review in some detail the new diagnostic tools allowing determination and quantitation of HCMV infection in blood of transplanted and AIDS patients. STUDY DESIGN: The determination and quantitation as well as the clinical significance of antigenemia, viremia, HCMV-infected circulating endothelial cells (CEC) and DNAemia will be discussed in view of the therapeutic management of HCMV disease. Levels of viremia represent the number of p72-positive cultured fibroblasts inoculated with 2 x 10(5)PBL, while levels of antigenemia represent number of pp65-positive PBL/2 x 10(5) PBL examined. The number of CEC is determined simultaneously and in parallel with antigenemia. DNAemia, both qualitative and quantitative, can be determined by polymerase chain reaction (PCR) per 1 x 10(5)PBL. The clinical utility of determining either immediate-early or late mRNA is still debated. RESULTS: In solid organ transplant recipients mean levels of viremia of 100 and of antigenemia of 400 correlate with onset of clinical symptoms. The time between first HCMV positivity and the onset of symptoms (>/= 10 days), together with the observation that most patients with reactivated infection clear virus without treatment, allowed the establishment of an antigenemia cut-off of 100 for the initiation of treatment. On the other hand, seronegative recipients of solid organs from seropositive donors must be treated preemptively, i.e. at first appearance of HCMV positivity in blood. Due to the risk of early appearance of HCMV pneumonia, the same preemptive approach must be used in bone-marrow transplant recipients. In acquired immunodeficiency syndrome (AIDS) patients with HCMV infection/disease, general criteria for initiation of treatment are more difficult to establish and treatment must be maintained. CEC are detected only in untreated disseminated HCMV infections with organ involvement. Qualitative DNA determination is useful only in special cases, such as in aqueous or vitreous humor of AIDS patients with HCMV retinitis. Quantitative DNA levels obtained by PCR are much more helpful for diagnosing HCMV disease and establishing initiation of treatment. CONCLUSIONS: New diagnostic procedures currently ensure fine monitoring of HCMV infections/diseases and evaluation of the effect of specific antiviral treatment in the immunocompromised host.
Journal Article↗
Amino acids involved in distinguishing between monotypes of rotavirus G serotypes 2 and 4.
Neutralizing monoclonal antibodies (N-MAbs) to serotype G2 and G4 rotaviruses were used to study intraserotypic variation by selection and characterization of N-MAb-resistant antigenic variants and reaction of N-MAbs with prototype rotavirus strains. Two G2-specific N-MAbs reacted with G2 rotaviruses S2, DS-1, RV-5 and RV-6 but not with 1076. Sequence analysis of the gene encoding VP7 of 1076 virus showed that the differences in amino acid sequence between 1076 virus and the other G2 strains at position 147, 213 and 217 correlated with the loss of N-MAb reactivity. Rotavirus variant mutation mapping data suggested that the amino acid difference at position 213 was likely to be of greatest importance. Rotavirus 1076 was defined as monotype b within G2 strains, whereas S2, DS-1, RV-5 and RV-6 belong to monotype a. The molecular basis for G4 subtypes/monotypes was also studied. The monotype G4b N-MAb 3A3 selected an antigenic variant with an amino acid mutation at position 96, whereas variants of the G4a-reactive N-MAb ST-3:1 showed a mutation at position 94, which produced a new, utilized glycosylation site. Neutralization by N-MAb ST-3:1 was also affected by amino acid changes at position 96. Reactions with these N-MAbs show that serotype G2 viruses can be divided into monotypes and confirm the observation that serotype G4 rotaviruses can be subdivided into subtypes/monotypes a and b. The G2 monotypes relate to differences at particular amino acids within antigenic region C and possibly region B, whereas antigenic region A is most important for G4 monotype differentiation.
Amino Acid Sequence↗
Human cytomegalovirus pp65 lower matrix phosphoprotein harbours two transplantable nuclear localization signals.
Human cytomegalovirus phosphoprotein pp65 is targeted to the cell nucleus immediately after infection. Deletion and point mutation analysis of the pp65 gene expressed in insect cells showed that two hydrophilic regions (HP1 and HP2) within the pp65 C-terminal 40% each harboured an independent nuclear localization signal (NLS); strong association to the nuclear stroma also requires the N-terminal domain. Either region, when fused to chloramphenicol acetyltransferase, localized the reporter protein to the nucleus in insect cells as well as in NIH 3T3 cells and human lung fibroblasts. In addition, HP1 was found to be the target of pp65 Ser/Thr phosphorylation in insect cells and a prokaryotically expressed HP1 was actively phosphorylated in vitro by casein kinase II, for which two site clusters map in HP1. These findings indicate that pp65 includes two NLSs, one of which has the potential to be modulated by phosphorylation.
3T3 Cells↗
Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS.
Three human cytomegalovirus (HCMV) strains (VR4760, VR4955, and VR5120) showing double resistance to ganciclovir (GCV) and foscarnet (PFA) were isolated from three patients with AIDS who underwent multiple sequential courses of therapy with GCV and PFA (A. Sarasini, F. Baldanti, M. Furione, E. Percivalle, R. Brerra, M. Barbi, and G. Gerna, J. Med. Virol., 47:237-244, 1995). We previously demonstrated that the three strains were genetically unrelated and that each of them was present as a single viral population in vivo. Thus, in each of the three cases, a single viral strain was resistant to both GCV and PFA. In the present paper, we report the characterization of the molecular bases of the double resistance and demonstrate that the PFA resistance is associated with a slower replication of HCMV strains in cell cultures. Sequencing of the UL97 and UL54 genes, GCV anabolism assays, and marker transfer experiments showed that GCV resistance was due to single amino acid changes in the UL97 gene product (VR4760, Met-460 --> Ile; VR4955, Ala-594 --> Val; VR5120, Leu595 --> Ser), while single amino acid changes in domain II of the DNA polymerase (VR4760 and VR5120, Val-715 --> Met; VR4955, Thr-700 --> Ala) were responsible for both the PFA resistance and the slow-growth phenotype. Thus, in these three cases, double resistance to GCV and PFA was not due to a single mutation conferring cross-resistance or to the presence of a mixture of strains with different drug susceptibilities. The HCMV DNA polymerase recombinant strains carrying the mutations conferring PFA resistance were sensitive to GCV and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). In addition, the same UL54 mutations were responsible for the slow growth of the clinical isolates, since the recombinant strains showed a marked delay in immediate-early antigen plaque formation and a reduction of infectious virus yield compared with AD169, from which they were derived. These results may have some important implications for the successful isolation, propagation, and characterization of PFA-resistant strains from clinical samples containing mixed viral populations.
AIDS-Related Opportunistic Infections↗
Clinical and virological monitoring of human cytomegalovirus infection in 294 heart transplant recipients.
Two hundred and ninety-four heart transplant recipients (HTR) were followed prospectively for a mean of 44.9 +/- 28.4 (range 1.0-100.8 months) after transplantation (tx). Immunosuppression was based on cyclosporine, azathioprine, and steroids, supplemented by a 7-day course of antithymocyte globulins. All patients were virologically monitored by inoculating aliquots of 2 x 10(5) peripheral blood polymorphonuclear leukocytes (PMNs) onto human embryonic lung fibroblasts monolayers grown in shell vials for early cytomegalovirus (CMV) identification and quantification (viremia). The same number of PMNs was cytocentrifuged onto glass slides for direct CMV pp65 antigen detection and quantification (antigenemia). Heparinized blood samples were collected weekly during the first 3 months following tx and at least twice a week if antigenemia and viremia levels were increasing. After 3 months, samples were collected if antigenemia and viremia persisted or when clinically indicated. The overall incidence of CMV infection was 53.4% (157/294). Only 32.4% (51/157) of the viremic patients required antiviral treatment because of symptomatic infection. Of the remaining 106 untreated CMV viremic HTR, 104 were asymptomatic while 2 had only mild clinical symptoms. The overall incidence of CMV infection in pre-tx CMV seropositive (CMV+) HTR was 50.9% (136/267); 75.7% (103/136) were asymptomatic and 24.3% (33/267); 75.7% (103/136) were asymptomatic and 24.3% (33/136) were symptomatic. The overall incidence of CMV infection in pre-tx CMV-seronegative (CMV-) HTR was 77.8% (21/27; P = 0.007 vs. seropositive HTR). Among 22 CMV- HTR with CMV+ donor, 20 (90.9%) had a CMV infection and all of them were symptomatic (versus 1 of 5 (20%) CMV- HTR with CMV- donor; P = 0.002, Fisher's exact test). The median numbers of circulating CMV-infected PMNs detected at the onset of clinical symptoms by the antigenemia and viremia assays were 385/2 x 10(5) and 100/2 x 10(5), respectively.
Adolescent↗
Double resistance to ganciclovir and foscarnet of four human cytomegalovirus strains recovered from AIDS patients.
Four human cytomegalovirus (HCMV) isolates from four different AIDS patients treated with both ganciclovir and foscarnet and not responding clinically to antiviral treatment, were studied in order to verify the occurrence of double resistance to both drugs, and to define whether single or multiple HCMV strains could be responsible for the double resistance. Peripheral blood leukocytes (PBL), the relevant conventional viral isolates, and plaque-purified strains from all four patients were examined by antiviral drug susceptibility testing by an immediate-early antigen plaque reduction assay and by restriction fragment length polymorphism (RFLP) analysis using polymerase chain reaction (PCR)-amplified multiple genome regions and endonucleases. All four HCMV strains had a high level of resistance to both ganciclovir and foscarnet. A single HCMV strain was shown to be responsible for the dual resistance in each patient. HCMV strain identity and uniqueness were shown for each of the four patients in blood samples, viral isolates, and plaque-purified strains. In addition, in two patients the same single HCMV strain shifted progressively from drug sensitivity to ganciclovir and then to ganciclovir-foscarnet resistance. These findings document that resistance to both ganciclovir and foscarnet of HCMV strains recovered from blood of AIDS patients represents an emerging problem. Although it is known that multiple HCMV strains may cocirculate in the blood of AIDS patients, single strains appear to be responsible for the dual resistance. Molecular mechanisms responsible for the double resistance of the four reported strains are under study.
AIDS-Related Opportunistic Infections↗
Polymerase chain reaction for prenatal diagnosis of congenital human cytomegalovirus infection.
The reliability of the polymerase chain reaction (PCR) for prenatal diagnosis of human cytomegalovirus (HCMV) infection was determined by retrospective testing of 35 amniotic fluids identified previously as positive or negative for HCMV by virus isolation. Amniocentesis was performed in 26 pregnant women with primary HCMV infection at 14-36 weeks gestation, 3-21 weeks after maternal infection. Blood samples were obtained from 20 fetuses for IgM determination and/or virus isolation. Amniotic fluid culture led to antenatal diagnosis of HCMV in 9 of the 13 infected fetuses (sensitivity 69.2%) with one case diagnosed at a second sampling. PCR was able to detect one additional infected fetus (10/13, sensitivity 76.9%). Nested PCR did not increase sensitivity of prenatal diagnosis. Three cases were not diagnosed by all the techniques employed. The specificity of virus isolation from and DNA detection by PCR in amniotic fluid was 100%. The negative predictive value for virus isolation from amniotic fluid was 76.5% and for DNA detection by PCR 81.2%, whereas the positive predictive value was 100% for both techniques. The results showed that neither approach can detect all cases of congenital HCMV infection prenatally, and that the time interval between maternal infection and sampling seems to be a major factor affecting the reliability of prenatal diagnosis.
Amniotic Fluid↗
Quantitation of human cytomegalovirus DNA in bone marrow transplant recipients.
HCMV DNA was retrospectively quantitated in the early post-transplant period in 36 paediatric bone marrow transplant (BMT) recipients prospectively monitored for human cytomegalovirus (HCMV) infection on the basis of antigenaemia and viraemia assays. Viral DNA was quantitated in peripheral blood leucocytes (PBL) by PCR using an internal control of amplification and a series of external standards. Densitometric analysis of hybridization results obtained on PCR products enabled construction of a standard curve from which DNA amounts of clinical samples, expressed in terms of genome equivalents (GE), were interpolated. Of the 36 BMT recipients, three had clinically symptomatic HCMV infection with mean peak levels of viral DNA > 5000 GE (antigenaemia and viraemia mean peak levels were 873 and 35, respectively), whereas 19 with HCMV reactivation were asymptomatic (five of them had abortive HCMV infection) showing mean peak DNA levels of 131 GE (and of 6.8 and 1.3 for antigenaemia and viraemia, respectively) (P < or = 0.01). Single or multiple courses of pre-emptive therapy with ganciclovir or foscarnet were given to 14/19 asymptomatic children in whom antigenaemia levels were > 2 or lower yet persisting. Overall, in the 14 asymptomatic treated patients the mean antigenaemia level was 9.3 (range 1-22), and the mean DNA level was 184.6 (range 20-710) GE. Antiviral drugs were also administered to the three symptomatic patients who, due to late diagnosis of HCMV infection, escaped preemptive therapy. Antiviral treatment caused marked decrease or disappearance of viral DNA, antigenaemia and viraemia in both symptomatic and asymptomatic patients. In conclusion, our study suggests that: (i) starting therapy in the presence of a mean antigenaemia level of 9.3 (range 1-22) corresponding to a mean DNA level of 184.6 (range 20-710) GE avoided occurrence of any major HCMV-related clinical complication; (ii) clinical symptoms were associated with antigenaemia levels > 100 and DNA levels > 1000 GE; (iii) the effect of antiviral treatment could be more carefully monitored by quantitation of viral DNA.
Adolescent↗
Ganciclovir therapy of congenital human cytomegalovirus hepatitis.
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Cytomegalovirus Infections↗
Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples.
A rapid screening assay for the detection of resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples was developed by using single doses of both drugs and an immediate-early antigen plaque reduction assay. Results of the rapid assay with peripheral blood leukocytes as the viral inoculum were compared with those of a conventional assay with cell-free virus from the relevant viral isolates. Both assays gave overlapping results. The rapid assay offers the following major advantages: (i) it provides results within 4 to 6 days, (ii) it can be performed with peripheral blood leukocytes, and (iii) it reliably detects drug-resistant human cytomegalovirus strains.
Antigens, Viral↗
A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate.
Multiple human cytomegalovirus (HCMV) strains frequently coexist in patients with AIDS, and chronic ganciclovir treatment may favor the emergence of ganciclovir-resistant viral mutants. We report the molecular and biochemical characterization of a HCMV ganciclovir-resistant strain (VR3480) previously recovered from a patient with AIDS who was undergoing multiple courses of ganciclovir treatment (G. Gerna, F. Baldanti, M. Zavattoni, A. Sarasini, E. Percivalle, and M. G. Revello, Antiviral Res. 19:333-345, 1992). Ganciclovir resistance of strain VR3480 was related to impaired ability to monophosphorylate the drug, as indicated by the finding that ganciclovir phosphorylation values for VR3480 were 30% of those shown by the HCMV reference strain AD169 in an in vitro activity assay. Sequencing of the UL97 gene of VR3480, which encodes the viral kinase responsible for ganciclovir phosphorylation, showed an in-frame deletion of three nucleotides resulting in the loss of a leucine at position 595 in the polypeptide. Mutant VR3480 UL97 DNA was able to transfer resistance to the AD169 strain in marker rescue experiments. Analysis of virus isolates and blood polymorphonuclear leukocyte samples spanning the 2-year follow-up period of the patient showed that ganciclovir-resistant strain VR3480 arose ex novo during prolonged antiviral treatment and accounted for the majority of virus load circulating in blood during the period of clinical resistance to ganciclovir treatment.
Acquired Immunodeficiency Syndrome↗
Herpes simplex virus-specific human cytotoxic T-cell colonies expressing either gamma delta or alpha beta T-cell receptor: role of accessory molecules on HLA-unrestricted killing of virus-infected targets.
Previous studies have demonstrated that herpes simplex virus-1 (HSV-1)-infected mononuclear cells are able to stimulate autologous peripheral blood mononuclear cells (PBMC) of immune donors and to activate HSV-specific cytotoxic T lymphocytes (CTL) expressing either gamma delta or alpha beta T-cell receptors (TCR). In the present report characterization of 10 gamma delta+ and six alpha beta+ HSV-specific cytotoxic T-cell colonies (TCC) is described. Cytotoxic colonies were derived from HSV-induced cell lines of three donors who, in previous experiments, had shown a prevalence of gamma delta+ or alpha beta+ effector cells. HSV-1 induced cell lines obtained from gamma delta responders included more than 80% of cells expressing V gamma 9/delta 2 TCR V region chains. gamma delta+ TCC also expressed V gamma 9/delta 2 molecules. alpha beta+ TCC all expressed CD8 antigen, while only one of 10 gamma delta+ TCC was CD8+, the others being CD4/CD8-double negative. The cytotoxic response of HSV-specific TCC was HLA-unrestricted; nevertheless CD8+ TCC were dependent on the expression of HLA class I on the surface of target cells to mediate cytolytic activity, while CD8- TCC were not. Blocking experiments with monoclonal antibody (mAb) specific for lymphocyte function-associated antigen-1 (LFA-1), which is expressed on all TCC, demonstrated that all alpha beta+ TCC and some gamma delta+ TCC also needed the interaction between LFA-1 and its ligands to develop cytotoxic activity. Altogether our data suggest that HSV-specific CTL may represent a population selected by a high concentration of antigen with a broad range of TCR affinities, which may play an important role as a first line of defence against HSV infection.
Antibodies, Monoclonal↗
Four dually resistant human cytomegalovirus strains from AIDS patients: single mutations in UL97 and UL54 open reading frames are responsible for ganciclovir- and foscarnet-specific resistance, respectively.
Four human cytomegalovirus strains with double resistance to both ganciclovir and foscarnet were recovered from acquired immunodeficiency syndrome (AIDS) patients. The four isolates were genetically unrelated and each of them consisted of a single viral population. In addition, for each isolate, UL97 and UL54 open reading frames sequencing showed a single aminoacid change in a conserved domain of each of the two genes.
AIDS-Related Opportunistic Infections↗
Detection of human cytomegalovirus DNA: how, when and where?
Although several methods have been utilized for the detection and quantification of human cytomegalovirus (HCMV) DNA, all of them can be divided into three groups: (i) detection of HCMV DNA directly in tissues by in situ hybridization or in situ polymerase chain reaction (PCR); (ii) detection of HCMV DNA in cell or tissue lysates by hybridization with DNA or RNA probes differently labelled-labels were progressively modified in order to provide an increasing sensitivity (hybridization products were revealed by radioactive, colorimetric or chemiluminescent procedures); (iii) detection of HCMV DNA in cell or tissue lysates by qualitative (single-step and nested) and quantitative (semiquantitative, competitive or noncompetitive) PCR. The selection of the methods to be employed depends primarily on the clinical situation which must be evaluated. Clinical samples for HCMV genome detection must vary accordingly.
Blotting, Southern↗
Prenatal diagnosis of congenital human cytomegalovirus infection.
Fifteen fetuses at risk of congenital human cytomegalovirus (HCMV) infection underwent prenatal diagnosis at 16-30 weeks' gestation by a combination of amniocentesis and fetal blood sampling. HCMV was isolated from the amniotic fluid in six patients, but HCMV-specific IgM was detected in only three of them. Two of the nine neonates, who were delivered following a negative prenatal diagnosis, had congenital HCMV infection diagnosed by virus isolation in the urine. The interval from infection to prenatal testing was 3 and 4 weeks in the two false-negative cases and > or = 7 weeks in the true-positive cases. Although timely testing for HCMV infection allows the option of termination of pregnancy, it may be flawed by false-negative results.
Amniocentesis↗